Peptidyl-tRNA dissociates from ribosomes of Escherichia coli during protein synthesis. If a mutant E. coli (pth) with temperature-sensitive peptidyl-tRNA hydrolase is placed at non-permissive temperatures, peptidyl-tRNA accumulates, protein systhesis is inhibited and the cells die, unable to form a colony when replaced at permissive temperatures. Because peptidyl-tRNA hydrolase is ubiquitous, its substrate and the processes which generate its substrate are probably universal characteristics of protein synthesis. This project proposes to exploit the E. coli pth strain to learn more about the processes that control the dissociation of peptidyl-tRNA. Physiological and pharmacological agents, as well as mutations, will be used to perturb the processes. The methodology will include measuring the properties and products of protein synthesis by cell-free extracts from genetically defined E. coli, isolation and characterization of peptidyl-tRNA that accumulates in pth strains and assaying the survival of the pth and various recombiannt strains at high temperatures. The ribosome editing hypothesis, concerning the fidelity of translation of genetic information, will be tested. The dissociation of peptidyl-tRNA from ribosomes is hypothesized to be the result of scrutiny of peptidyl-tRNA by ribosomes and preferential ejection of those peptidyl-tRNAs whose structure does not correctly complement the mRNA.